As was summarized previously in this and the related NO annual report (DK 025093-18) over the last two decades we have, with our immediate and international collaborators, shown a major new route for NO formation in the mammalian and human bodies, i.e., the reduction of nitrite ions to form NO. This complements, especially under hypoxic conditions, the activity of the various NOS enzymes. As a result we have focused on physiological and pharmacological effects of nitrite administration, as well as nitrate ingestion (which is converted to nitrite by salivary bacteria) and the fluxes in these nitrogen oxides in tissues and in the mammalian body. As summarized in the related report, various physiological pathways such as brain blood flow, red cell storage, platelet reactivity and blood clotting are very much influenced by the levels of these compounds. Recent work summarized in the DK 025093 series of reports suggest that some mammalian tissues can also reduce nitrate ions to nitrite and then NO. It is thus essential that we establish the utility and safety of ingestion of these compounds in foods and medicines and to examine how various factors affect their absorption from the diet. We also wish to measure their levels in various biological fluids, such as blood and urine, as well as in the environment. For some years we have been working with a group in Lisbon to develop electrode-based assays for nitrite in water and other environmental fluids; in view of the importance of nitrite in blood and other biological fluids we have now redirected this work to a more micro scale so that measurements on animal and human samples may be feasible and have recently published details of a micro-electode which may allow such assays at physiologically relevant concentrations. Detailed metabolic studies of nitrate-nitrite-NO metabolism in animals and people require tracer studies which may be approached using non-toxic heavy isotopes. Our collaborators in Newcastle are developing protocols to do this in animals and then perhaps in human subjects. The have sent us samples from various animal ingestion studies and we are using our highly sensitive and accurate chemi-luminesence methods to quantity nitrate and nitrite levels in these blood samples. (One of their staff visited our laboratory for several weeks to learn how to do these assays.) Our collaborators in Sao Paulo, Brazil have much experience in pharmacology of NO and we are working with them on various animal models of hypertension with the hope of eventual clinical studies. Our first results, recently published, show that administration of an anti-oxidant Tempol to rodents with reno-vascular hypertension improved the vascular responses to nitrite but that the anti-hypertensive responses were not affected. Lastly we have established a collaboration with a muscle physiology group in Exeter, UK and we anticipate -once all permissions are effected -measuring the levels of nitrate at rest and with exercise - in the human tissues to see if our animal results obtain for human subjects. We have also participated in an NIH-sponsored discussion of the utility and safety of orally administered nitrate and nitrite in food and as medications. These deliberations were recently published and suggest many possible health benefits of increasing nitrate and nitrite levels, especially by oral administration, and suggest new studies to see if previously postulated harmful effects of such ingestion were really valid.